A novel method for DNA molecular counting.

K Hirano; T Fukami

doi:10.1093/nass/44.1.157

Home/Archive/PubMed

PubMed · 12903316

A novel method for DNA molecular counting.

K Hirano·T Fukami

View original source ↗Export metadata ↓

Abstract

We have developed a new method for counting DNA molecules using 'capillary-plates' consisting of a large number of small glass-capillary 'channels' fused together in parallel. PCR mixtures containing serially diluted DNA templates with the DNA indicator dye Hoechst 33258 were poured into the plates and sealed with silicone rubber-plates. Following 40 PCR cycles, fluorescence microscopy revealed that the fluorescence in some channels had increased about three-times more than in others at template concentrations of 1 fM or lower. No bright fluorescence was observed in the absence of template. The relationship between the proportion of fluorescent channels in the capillary-plates and the template concentrations was linear according to Poisson probabilities in the range of 0.1-1,000 aM. These results demonstrate the amplification of single templates in the channels, and that a small number of templates could be quantified by counting the proportion of positive channels on the capillary-plates.

Explore related subjects

Bisbenzimidazole DNA Fluorescent Dyes Microscopy, Fluorescence Nucleic Acid Amplification Techniques Polymerase Chain Reaction Journal Article Research Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

K Hirano, T Fukami. 2000. A novel method for DNA molecular counting.. https://doi.org/10.1093/nass%2F44.1.157

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Studies on the interactions between DNA and PAMAM with fluorescent probe [Ru(phen)2 dppz]2+.

The interactions between plasmid DNA and cationic polymers are of interest for their potential biological applications. In this paper, the interactions of DNA with polyamidoamine (PAMAM) dendrimer were studied by fluorescence spectroscopy and stopped-flow technique. A rapid and reproducible fluorescent assay method had been developed for assessing PAMAM and DNA interactions using [Ru(phen)2 dppz]2+ as a probe. We further studied the kinetics of PAMAM binding to DNA and the reverse process of DNA dissociation from the complexes. The results indicated that DNA condensation was the rate-determining step during the complexation process, while DNA unfolding and expansion was the rate-determining step during the DNA dissociation process. At N/P ratios before reaching the thermodynamically most stable state, the complexes of DNA and PAMAM were incompact and could dissociate to some extent. And at N/P ratios above 2.0, DNA was fully condensed by PAMAM and dissociation was increasingly difficult. These results provided some useful instructions for self-assembling and disassembling of DNA as well as efficient gene delivery applications.

Bisbenzimidazole↗

PubMed2006

Simultaneous quantitative measurement of luciferase reporter activity and cell number in two- and three-dimensional cultures of hepatitis C virus replicons.

Hepatitis C virus (HCV) is a global health problem and an important human pathogen. The development of cell culture models for HCV infection has been difficult to accomplish, primarily because HCV is very sensitive to the host cell state. Future models will require the use of three-dimensional (3D) cultures that model the host cell state and environment more accurately. Higher information content screens for anti-HCV therapeutics will also involve 3D cell cultures. Here we report a method for screening cell models for HCV replication that involves normalizing luciferase reporter activity based on cell number in two-dimensional (2D) and 3D HCV replicon cultures. Human hepatoma cells stably replicating luciferase-containing HCV replicons were cultured in 2D monolayer culture and 3D spheroid culture. Optimization of cell lysis was performed so that cell lysates could be used to quantify both luciferase activity and cellular DNA content. Cellular DNA content was quantified using Hoechst 33258 dye and was converted to cell number. The method is straightforward, reproducible, and sensitive down to 5000 cells. This method enables low-throughput but high-information content screening of HCV replicons, with the potential for high-throughput screening in a variety of 3D cultures and cocultures.

Bisbenzimidazole↗

PubMed2005

Assay for the quantification of small-sized fragmented genomic DNA.

This paper presents a simple assay for the direct quantification of small-sized (up to 1000 bp) fragmented DNA based on the differential separation between large- and small-sized DNA by polyethylene glycol precipitation. The assay can be applied to as low as 1 microg ml(-1) DNA and has a very low DNA detection limit (0.01 microg ml(-1) fragmented DNA), which can be further lowered at least 10-fold with anion exchange chromatography. The assay quantifies for the first time a more direct indicator of the extent of DNA damage than the usually assessed DNA nicks and base modifications, since small-sized fragmented DNA represents an unrepairable DNA damage of necrotic and apoptotic events that are related to normal and abnormal biological conditions.

Bisbenzimidazole↗